Quantification of Bile Acids by UPLC/MS/MS

Serum BA extraction and quantification were described previously (15 ). Liver BA concentrations were quantified by a recent method using ultra performance liquid chromatography- tandem mass spectrometry (UPLC/MS/MS) (14 (link)). BA stock solutions were diluted with 50% methanol and spiked with internal standards (²H₄-GCDCA and ²H₄-CDCA) to construct standard curves between 5 and 20,000 ng/ml. All standard curves were constructed using a 1/concentration² weighted quadratic regression, and the correlation coefficient (r²) for all BAs was above 0.99. The limit of detection (signal/noise ratio=3) for the various BAs was in the range of 5-10 ng/ml, which equals 0.01-0.02 nmol/ml. For a preliminary analysis of hydroxylated BA, we obtained taurine-conjugated and unconjugated 3α,6α,7α,12α-tetrahydroxyl BA from Dr. Mary Vore (University of Kentucky, Lexington, KY). We optimized and obtained the multiple reaction monitoring (MRM) conditions for these two standards. Then we used the same MRM conditions to obtain chromatographs of tetrahydroxy BAs in BDL samples.